M ATERIALS AND M ETHODS Materials

Materials

Seeds of mung bean (Vigna radiata), kidney bean (Phaseolus vulgaris), soybean (Glycine max), white lupine (Lupinus albus), blue lupine (L. angustifolius), yellow lupine (L. luteus) and peanut (Arachis hypogaea) were purchased from Vreeken’s Zaden (Dordrecht, The Netherlands). Daidzein, genistein, trans-resveratrol and soyasaponin Bb were purchased from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile (ACN; ULC/MS grade), water acidified with 0.1% (v/v) acetic acid (HOAc) or 0.1% (v/v) formic acid (FA) (ULC/MS grade), and methanol (MeOH) (ULC/MS grade) were purchased from Biosolve (Valkenswaard, The Netherlands). Water for purposes other than UHPLC was prepared using a Milli-Q water purification system (Millipore, Molsheim, France). Growth media, Bacto brain heart infusion (BHI) broth was purchased from BD (Franklin Lakes, NJ, USA);

tryptone soya broth (TSB) and agar bacteriological from Oxoid Ltd (Basingstoke, UK) and peptone physiological salt solution (PPS) from Tritium Microbiologie (Eindhoven, The Netherlands). All other chemicals were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich.

Microorganisms

Food grade fungi Rhizopus oryzae (LU 581) and Rhizopus oligosporus (LU 575) (Laboratory of Food Microbiology, Wageningen University, Wageningen, The Netherlands), were used to elicit the legume seeds during germination. L.

monocytogenes EGD-e and methicillin-resistant Staphylococcus aureus 18HN (MRSA, kindly provided by RIVM, Bilthoven, The Netherlands) were used to test the antimicrobial properties of the extracts from legume seedlings. Glycerol stocks (50% (v/v)) of these microorganisms were kept at -80 °C.

Elicitation and extraction of legume seedlings

Legume seeds were germinated in the presence of Rhizopus sp. as previously described.

[16] Briefly, legume seeds were soaked in water for 1 day, germinated for 2 days at 25 °C (100% RH), and subsequently elicited with the fungus (approx. 1.5 x 10⁷ CFU/g seed) for 5 days at 30 °C (55-85% RH). After the elicitation period, seedlings were freeze-dried, milled, defatted with hexane and extracted with 80% (v/v) aqueous MeOH, as described elsewhere.^[16] The MeOH extract obtained from the seedling meal was cleaned from the presence of sugars and other water-soluble components by solid-phase extraction (SPE) with Sep-Pak Vac C18 cartridges (Waters, Milford, MA, USA), following the protocol of the manufacturer. The final SPE-cleaned seedling extract was dried with a flow of nitrogen gas, solubilized in tert-butanol and freeze-dried to obtain a powder. All extracts were kept at -20 °C until further analysis.

Compositional analysis by RP-UHPLC-PDA-ESI-MS

Compositional analysis was performed on an Accela ultra high performance liquid chromatography (RP-UHPLC) system (Thermo Scientific, San Jose, CA, USA) equipped with a pump, autosampler, photodiode array (PDA) detector and ESI-ion trap mass spectrometer (MS).

For flavonoid, isoflavonoid and saponin analysis, seedling extracts (2 µL; 5 mg/mL in MeOH) were injected onto an Acquity UPLC BEH Shield RP18 column (2.1 mm i.d. x 150 mm, 1.7 µm particle size) with an Acquity UPLC Shield RP18 Vanguard guard-column (2.1 mm i.d. x 5 mm, 1.7 µm particle size; Waters, Milford, MA, USA). Water containing 0.1% (v/v) HOAc and 1% (v/v) ACN, eluent A, and ACN containing 0.1%

(v/v) HOAc, eluent B, were used as solvents at a flow rate of 300 µL/min. The following elution gradient was used: 0-1 min isocratic on 9% (v/v) B; 1-2.5 min, linear gradient from 9 to 25% B; 2.5-9.5 min, linear gradient from 25 to 50% B; 9.5-12.5 min isocratic at 50% B; 12.5-22.5 min, linear gradient from 50 to 100% B; 22.5-24.5 min isocratic at 100% B; 24.5-25 min, linear gradient from 100 to 9% B; 25-30 min, isocratic at 9% B.

Column temperature was set at 35 °C and the PDA detector was set to measure from 200-600 nm.

For stilbenoid analysis, seedling extracts (2 µL, 5 mg/mL in MeOH) were injected onto a Hypersil Gold C18 column (2.1 mm i.d. x 150 mm, 1.9 µm particle size, Thermo Scientific, San Jose, CA, USA). Water containing 0.1% (v/v) FA + 1% (v/v) ACN, eluent A, and MeOH containing 0.1% (v/v) FA, eluent B, were used as solvents at a flow rate of 300 µL/min. The following elution gradient was used: 0-1 min isocratic on 0% B;

1-2 min linear gradient from 0 to 30% B; 2-18 min linear gradient from 30 to 80% B;

18-23 min linear gradient from 80 to 95% B; 23-24 min linear gradient 95 to 100% B;

24-26 min, linear gradient from 100 to 0% B; 26-31 min, isocratic on 0% B. Column temperature was set at 40 °C and PDA detector was set to measure from 200-600 nm.

MS analysis was performed on a LTQ Velos (Thermo Scientific, San Jose, CA, USA) equipped with a heated ESI-MS probe coupled to RP-UHPLC. Spectra were acquired over an m/z (mass to charge ratio) range of 150−1500 Da in both positive (PI) and negative (NI) mode. Data-dependent MSⁿ analysis was performed on the most intense (product) ion with normalized collision energy of 35%. A dynamic mass exclusion approach was used, in which a compound detected five times as most intense was subsequently excluded for the following 10 s, allowing data dependent MS² of less intense co-eluting compounds. The system was tuned with genistein and resveratrol in PI and NI mode via automatic tuning using Tune Plus (Xcalibur v.2.2, Thermo Scientific).

Nitrogen was used as sheath and auxiliary gas. The ITT temperature was 400 °C and the source voltage was 3.50 kV (NI) or 4.50 kV (PI).

Tentative annotation of phytochemicals

Compounds were tentatively annotated based on UV-Vis and MS spectral data, obtained by means of Xcalibur (v.2.2, Thermo Scientific), using the approach reported earlier on legume seeds. A summary of the different diagnostic UV-Vis absorption and MS fragmentation patterns is provided in Table S2.1 (supplementary data). Briefly, UV-Vis absorption spectra and retro-Diels-Alders (RDA) fragmentation patterns were used to indicate the subclass of phenolic compound (isoflavonoid, flavonoid, stilbenoid, phenolic acid).^[19,20] Full MS and tandem MS scans provided information regarding molecular weight and substitutions of the phenolic skeleton by means of characteristic neutral losses. The configuration of the prenyl group (chain or ring-closed) attached to the skeleton was determined by typical neutral losses in MS² PI: a neutral loss of 56 Da (C₄H₈) was used to distinguish a prenyl chain, whereas a ring-closed prenyl typically showed neutral losses of 42 Da (C₃H₆), 54 Da (C₄H₆), 60 Da (C₃H₆ + H₂O) and 15 Da (CH₃·).^[21,22] The position of the prenyl group within the phenolic skeleton (i.e. A- or B-ring) was elucidated by analysis of the retro-Diels-Alders (RDA) fragments^[23] in PI: when the C-ring of (iso)flavonoids was cleaved in MS³, one of the remaining fragments still contained one carbon reminiscent of the prenyl chain (split in MS²), which can be used to diagnose the ring at which the prenyl was attached.^[22] The position of the prenyl group within the A-ring was established based on a study using authentic standards to distinguish between C6 and C8 chain prenylated isoflavone isomers by MS fragmentation pattern.^[22] The fragment ion in MS³ PI [M+H-C₄H₈-C₂H₂O]⁺ was

diagnostic for C8 prenylation, whereas the fragment ion in MS³ PI [M+H- C₄H₈-CO]⁺ was diagnostic for C6 prenylation. Furthermore, previous tentative MS annotation of prenylated pterocarpans^[24] was confirmed by means of NMR spectroscopy of the purified molecules.^[10]

Saponins were distinguished from phenolic compounds in the seedling extracts during the same UHPLC run as saponins (i) do not show any or little UV absorbance contrary to phenolics, except for DDMP-conjugated saponins, which have maximum UV absorption at 295 nm, (ii) have higher molecular weights than most phenolics due to their larger carbon skeleton and more extensive glycosylation (2-5 sugar units; mass range 800-1500 Da), and (iii) are usually more apolar than phenolics due to their triterpenoid backbone. For the tentative annotation of saponins the presence of m/z values of the triterpenoid aglycones, such as sapogenol B (m/z 457), sapogenol A (m/z 473) and sapogenol E (m/z 455) in MS² or MS³, was used as a diagnostic tool.^[25]

Furthermore, commonly observed neutral losses were those of H₂O, CO₂ and individual sugars (132 Da for a pentosyl unit, 146 Da for a deoxyhexosyl unit, 162 Da for hexosyl unit, 176 Da for a hexuronyl unit) resulting from cleavage of the glycosidic linkages.^[26]

Quantification of phytochemicals

Quantification of phenolics was based on the UV absorption at 260 nm (white, yellow and blue lupine), 280 nm (mung bean, kidney bean and soybean) and 315 nm (peanut). In case of co-elution, the UV peak area was divided over the co-eluting compounds in a ratio based on the MS intensity. Quantification of phenolic compounds was performed using the following equation (Eq. 2.1),^[27] derived from the Lambert-Beer’s law:

𝐶 [𝑀] =

𝑖𝑛𝑗 ×𝑘_{𝑐𝑒𝑙𝑙}

in which C (M) is concentration, area is the integrated area of the UV peak at the specific wavelength (AU·s), Q is the flow rate (5 µL/s), ε (AU/M·cm) is the molar extinction coefficient, l is the path length of the UV cell provided by the manufacturer (5 cm), Vinj is the injected volume of sample (2 µL), and k_cell is a constant related to the cell geometry of the UV detector ^[27]. This equation relates the duration of absorbance given by the UHPLC system (AU·s) to an actual absorbance value (AU) for the Lambert-Beer’s equation. The kcell represents the correction factor for the absorption of light by the coating material of the flow cell. The k_cell (0.82 ± 0.09) was determined with standard solutions of daidzein (248 nm), genistein (263 nm) and resveratrol (310 nm) (with five concentrations each, in the range of 0.001-0.1 mg/mL).

Because the ε of most tentatively annotated compounds were unavailable, for each subclass of phenolics (e.g. flavanone, flavone, flavonol, isoflavanone, isoflavone, coumestan, coumaronochromones, pterocarpan, stilbenoid, phenolic acid) a

representative compound was chosen and its ε reported in literature was used (corrected for the wavelength used in this work) (Table S2.2).

Because most saponins have little UV absorption, quantification of saponins was based on MS, using an external standard solution of soyasaponin Bb (0.001-0.1 mg/mL). Results are reported as soyasaponin Bb equivalents per gram of extract (Bb eq./g DW extract).

Antibacterial activity assay

Bacteria (L. monocytogenes EGD-e and MRSA 18HN) were streaked from a glycerol stock to a BHI agar plate and incubated 24 h at 37 °C, after which one colony was transferred to BHI broth (10 mL) and further incubated for 18 h at 37 °C. These overnight cultures were diluted 100,000 times with fresh TSB (final inoculum concentration 3.6 ± 0.5 Log₁₀CFU/mL). Stock solutions of the different seedling extracts were prepared in 70% (v/v) aqueous ethanol (EtOH). Equal volumes (100 µL) of bacteria and a series of 2-fold dilutions of seedling extracts (final concentrations tested 0.25-1.0 mg/mL) in TSB were mixed into a 96-well plate (maximum concentration of EtOH in test was 2% (v/v)). The 96-well plate was incubated in a SpectraMax PLUS 384 (Molecular Devices, Sunnyvale, CA, USA), at 37 °C with constant shaking, and the optical density (OD) at 600 nm was measured every 5 min for 24 h. Positive controls (ampicillin at 3.1 µg/mL in water for L. monocytogenes and vancomycin at 2.0 µg/mL in water for MRSA), negative controls (TSB suspension of bacteria with 2% (v/v) EtOH), and blanks (extracts and TSB medium with no bacteria) were considered for optical comparison and sterility control. The time to detection (TTD) was defined as the time to have a change in OD of 0.05 units.^[28] Extracts were tested in two independent biological duplicates.

When no change in OD was observed after the 24 h of the antimicrobial susceptibility test, cell viability was verified by plate counting. Briefly, 100 µL of the well with no change in OD was decimally diluted in PPS solution and 100 µL of each dilution was spread onto BHI agar plates. Plates were incubated for 24 h at 37 °C. The MBC was defined as the lowest concentration of seedling extract that resulted in >99% bacterial inactivation from the initial bacterial inoculum.

Time-dependent inhibition

An overnight bacterial culture was diluted to 10⁴ CFU/mL and mixed with the seedling extracts (final concentration 1.0 mg/mL), and incubated at 37 °C, 125 rpm. At different time points (0-6 h), 100 µL of culture medium were taken and decimally diluted in PPS.

Dilutions were spread on BHI agar plates and incubated overnight at 37 °C, after which colonies were counted.

Linear regression analysis

To investigate the relationship between phytochemical composition and antimicrobial activity of the seedling extracts, multiple linear regression (MLR) was used (Eq. 2.2):

𝑦 = 𝑦̂ + 𝛽

𝑥

+ 𝛽

𝑥

+ . . . +𝛽

𝑥

where y is the measured TTD of growth (indicative of their antibacterial activity) for the seedling extracts (extracts that showed TTD beyond the 24 h of the test cannot be included in the statistical analysis), ŷ is the TTD of the control experiment (TSB suspension of bacteria with 2% (v/v) EtOH), β is the coefficient of each independent variable x, and x is the concentration of each phytochemical class analysed (phenolic compounds and saponins) in the legume seedling extracts. Significance of the model and of the coefficients was evaluated by the t-test. Analysis was performed using IBM SPSS Statistic v.22 software (SPSS Inc., Chicago, IL, USA).

Prediction of chemical properties

LogP values were predicted using the weighted method (default settings) from the software Marvin (version 6.0.5, 2013, ChemAxon, Budapest, Hungary).

R

Content and composition of phenolic compounds in extracts

The tentative annotation of the peaks was performed based on a comparison of spectral data from LC-MS/MS (including retention time, UV spectra and fragmentation pattern) to those from literature (Table S2.1).[16,19,20,25,29-32] Overall, 177 different phenolic compounds were tentatively annotated in the legume seedling extracts. Table S2.3 shows the list of compounds with their respective MS fragmentation pattern in NI and PI mode.

The majority of phenolic compounds belonged to the isoflavonoid class, including 62 isoflavones, 23 isoflavanones, 9 pterocarpans, 5 coumestans, 4 coumaronochromones, 3 pterocarpenes, and 1 isoflavan. From the flavonoid class, 24 flavones, 10 flavanols and 5 flavanones were tentatively annotated. Furthermore, 25 stilbenoids, as well as 4 phenolic acids, were exclusively present in peanut. Lastly, 2 chromones were found exclusively in lupine species. In total, 57 phenolic compounds were prenylated and belonged to the isoflavonoid, stilbenoid and chromone classes.

The contents of phenolic compounds per extract of legume seedlings (G) and fungus-elicited seedlings (GF), grouped as non-prenylated glycosides (with glycosyl residues attached), non-prenylated aglycones (only hydroxyl or methoxyl groups attached), prenylated glycosides and prenylated aglycones (prenyl group attached), is given in Figure 2.1. The concentration of each individual component is given in Table S2.4. The total content of phenolics ranged from 35 to 212 mg/g DW extract. Blue and

yellow lupine (G and GF) were the legume extracts with the highest overall content of phenolic compounds. The peanut seedling extracts had the highest content of prenylated aglycone compounds (52 ± 3.6 mg/g DW G extract and 93 ± 2.9 mg/g DW GF extract), followed by soybean (30.1 ± 0.3 mg/g DW GF extract), kidney bean (25 ± 2.1 mg/g DW GF extract) and yellow lupine (19 ± 0.9 mg/g DW GF extract).

Figure 2.1. Content (mg/g DW extract) of non-prenylated glycosides, non-prenylated aglycones, prenylated glycosides and prenylated aglycone phenolic compounds in extracts from germinated legume seeds (G) and fungus-elicited (GF) legume seedlings.

The normalized weight distribution of the subclasses of the phenolics present in the legume seedling extracts is shown in Figure 2.2A. Except for peanut, 50 to 90%

(w/w) of the phenolic content belonged to the isoflavonoid class, and 5 to 50% (w/w) to the flavonoid class. With respect to the subclasses, the lupine species showed similar profiles among each other (mainly isoflavone and flavone). Mung bean and kidney bean also showed similarities in the subclasses of phenolics produced (e.g. isoflavanone and flavonol), as expected by the phylogenetic relation between these two species.^[19]

Soybean was the only seed containing high abundance of pterocarpans (when elicited), whereas peanut contained around 70% (w/w) stilbenoids. Figure 2.2B shows the normalized weight distribution of the subclasses concerning the prenylated compounds.

No prenylated flavonoids were present in the legume seedlings. Mung bean and kidney bean mainly contained prenylated isoflavanones; soybean mainly contained prenylated pterocarpans; the lupine species mainly contained prenylated isoflavones and peanut mainly contained prenylated stilbenoids.

Figure 2.2. Normalized weight distribution of phenolic (sub)classes for all compounds (A) and for prenylated compounds (B) found in the extracts of legume seedlings, belonging to the isoflavonoid class (green), flavonoid class (blue), stilbenoid class (red), phenolic acid class (orange) and chromone class (purple).

Further analysis of these prenylated compounds showed that chain prenylation (e.g. 3, 3-dimethylallyl) was the most abundant type of prenylation (Figure 2.3A). The mung bean and lupine extracts almost exclusively contained chain-prenylated molecules, whereas soybean contained around 80-90% (w/w) ring-closed prenylated molecules (e.g. 2, 2-dimethylpyran or 2’’-isopropenyl furan). Double chain-prenylated aglycones were found in white lupine GF seedling extract (lupichromone), blue lupine GF seedling extract (angustone A) and peanut G and GF seedling extracts (6 and arahypin-7) (Table S2.3). Regarding the mono-prenylated compounds (both chain and ring-closed prenylated), Figure 2.3B shows the normalized weight distribution of the different tentatively annotated prenylated positions in the main phenolic skeletons. For this, ring and atom numbering was adapted to facilitate comparison of different (sub)classes of phenolics.^[33] The position of the prenyl group can be tentatively annotated by means of diagnostic MS fragmentation patterns, as described in the section 2.5.^[22] Using these tools we found that prenylated compounds from mung and kidney bean were predominantly prenylated at the A-ring α position (α), whereas A-ring β position (β) was more predominant in the other legume species.

Figure 2.3. Type of prenylation (chain, double chain or ring-closed) (A) and tentative position of the prenyl group (B) of the prenylated phenolic compounds found in extracts from germinated seeds (G) and fungus-elicited (GF) legume seedlings.

Content and composition of saponins in extracts

The saponin content in legume seedlings ranged from 8-101 mg Bb eq./g DW extract (Figure 2.4). Tentatively annotated saponins are listed in Table S2.5 and individual contents are present in Table S2.6. In general, white lupine and peanut seedling extracts had the lowest contents of saponins, whereas soybean and blue lupine G extracts had the highest contents of saponins. Most abundant saponins found in all legume seedling extracts were from the oleanane class.^[5] More specifically, we found high abundance of sapogenol B-type saponins, such as soyasaponin Bb (also known as soyasaponin I, MW 942 Da), soyasaponin βg (DDMP-conjugated soyasaponin Bb, MW 1068 Da) and soyasaponin Be (MW 940 Da). In addition the soybean extracts also contained sapogenol A-type saponins, such as soyasaponin Aa (MW 1364 Da), soyasaponin Ab (MW 1436) and soyasaponin Ae (MW 1202). This result was consistent with a previous saponin screening in legume seeds.^[34]

Figure 2.4. Content (mg/g DW extract) of soyasaponins in extracts from untreated legume seeds (U), germinated seeds (G) and fungus-elicited (GF) legume seedlings. The quantified saponins (MW 800-1500 Da) represent ≥ 80% of all saponins present in the extracts.

Antibacterial activity of extracts

All seedling extracts were tested in the antimicrobial susceptibility test using foodborne L.

monocytogenes as target pathogen. The TTD of growth was determined for all G and GF seedling extracts at different concentrations (Figure 2.5A). The TTD increased with increasing concentration of the seedling extracts. The G seedling extracts delayed the growth of L. monocytogenes by 1 h (kidney bean) up to 7 h (yellow lupine) at the highest extract concentration tested (1.0 mg/mL), except for the peanut G, which delayed the growth for more than the period of the test (TTD > 24 h). The GF seedling extracts were more effective inhibitors than the G extracts. The most antimicrobial GF seedling extracts were those from peanut, mung bean, soybean and yellow lupine. For MRSA, the same type of assay was performed with the GF extracts, as these were the most active ones (Figure 2.5B). Only the peanut GF extract was active against MRSA at 0.5 and 1.0 mg/mL.

Verification of viability on plates revealed that 1.0 mg/mL of peanut G extract and 0.5 mg/mL of peanut GF extract were minimum bactericidal concentrations (MBC) against L. monocytogenes (Figure 2.5C), and 1.0 mg/mL of peanut GF extracts was

Figure 2.5. Time to detection (TTD) of growth of (A) L. monocytogenes and (B) MRSA 18HN in the presence of different seedling extracts at increasing concentrations (0.25, 0.50 and 1.0 mg/mL). Seedling extract (G, light grey), extract from fungus-elicited seedlings (GF, dark grey). The most active seedling extracts are shown in red colour. TTD of the control experiment for L. monocytogenes was 9.7 ± 0.8 h (inoculum 3.2 ± 0.4 Log10 CFU/mL) and for MRSA was 5.7 ± 0.2 h (inoculum 3.9 ± 0.3 Log10 CFU/mL). (C) L. monocytogenes and (D) MRSA cell counts after the 24 h of incubation with the most active seedling extracts. Dotted line represents the cell count detection limit (d.l.) (E) L. monocytogenes and (F) MRSA killing kinetics in the presence of the active peanut GF extract (1.0 mg/mL) in comparison with the negative control (TSB broth) and control antibiotics. Data are means of two independent biological duplicates with standard error bars.

To further study the rate of inhibition of the most antibacterial legume extracts during the first hours of exposure, the growth of L. monocytogenes and MRSA was monitored with the active GF extracts at 1.0 mg/mL (Figure 2.5E and 2.5F). Regardless the pathogen, the extract of peanut GF reduced the levels of bacteria to below detection limit (i.e. reduction > 99% from initial inoculum) at already 2 h of incubation. The control antibiotics, however, did not show a significant reduction in cell counts at 2 h. At 24 h, no cell colonies were detected for both the peanut GF extract and the control antibiotics.

Correlation between phytochemical composition and antibacterial properties of extracts

To study the effect of the different phytochemicals analysed on the antibacterial activity of the extracts, multiple linear regression was performed using the TTDL. monocytogenes as response variable. After considering the total content of saponins or phenolic compounds, and subsequently the content of the different types within these compound classes, as predictor variables (Table S2.7), the main phytochemical group determining the TTD of bacterial growth of the extracts from legume seedlings were the prenylated aglycones (p < 0.001, Figure 2.6A). The content of non-prenylated aglycones was the second variable influencing the TTD, but to a lesser extent.

Figure 2.6. Relation between the content of total prenylated aglycones (A) and β prenylated aglycones (B) with the time to detection of L. monocytogenes growth (n = 42).

Regarding the position of the prenyl group, the content of compounds prenylated at position β was highly correlated with antibacterial activity (Figure 2.6B, p <

10^-6 and R²adj 0.62), in comparison with the content of compounds prenylated at position α (R²_adj 0.27) and δ (R²_adj 0.48). For MRSA the content of prenylated aglycones significantly affected (p < 0.01) the TTD of bacterial growth and only prenylation at position β was significantly (p < 0.005) related to the antibacterial activity of the legume seedling extracts (Figure S2.1 and Table S2.7).

D

Prenylated phenolic aglycones significantly correlate with the antibacterial activity of extracts from legume seedlings

In this study, we performed an extensive characterization of phytochemicals of seven major crop and forage legumes, after germination of the seeds alone, and after subsequent elicitation by fungus. We tentatively annotated 177 compounds distributed over 13 phenolic subclasses. Taken into consideration this large structural variety, we found that the antibacterial activity of the extracts was significantly correlated with the content of prenylated phenolic aglycones (for L. monocytogenes and MRSA) and to a lesser extent (only for L. monocytogenes) to the non-prenylated aglycones (Figure 2.6A).

In accordance with our results, the antimicrobial activity of non-prenylated aglycones, such as daidzein and genistein, is known to be lower (MIC values between 100-1000 µg/mL)^[35] than those of prenylated analogues (MIC values below 100 µg/mL).^[13]

Prenylated phenolic glycosides and non-prenylated glycosides were not significantly correlated (p > 0.05) with the antimicrobial properties of the legume seedling extracts, as glycosylation is a strategy commonly used by plants to inactivate natural products for storage purposes.^[6]

Among the prenylated phenolic compounds, differences in the type of prenyl

Among the prenylated phenolic compounds, differences in the type of prenyl